Ras is a guanine nucleotide binding protein, and participates in signal transduction of cells. When a receptor of cells is activated, “GDP binding Ras” in cells becomes “GTP binding Ras”.
This “GTP binding Ras” is bound to “target proteins of Ras” such as Raf-1, B-Raf, RGL, Ral GDS, MEKK, P13KK and the like. These “target proteins of Ras” have a Ras binding domain (RBD) to which the GTP binding Ras can be bound, and the GTP binding Ras is bound to this domain of these “target proteins of Ras” to transmit necessary signals into cells.
Ras is a key protein of intracellular signal transduction, and the “target proteins of Ras”, such as Raf-1, are a center of the intracellular signal transduction system in which signals from Ras are transmitted according to the types.
Accordingly, a substance capable of specifically blocking the binding domain with the GTP binding Ras in the “target proteins of Ras”, if any, can specifically inhibit an intracellular signal transduction system by Ras, and it is useful to treat or prevent various diseases triggered by the signal transduction. For example, with respect to tumor cells, proliferation or differentiation of tumor cells can be inhibited by specifically controlling the signal transduction that induces proliferation or differentiation with the “target cells of Ras” to treat cancers or inhibit metastasis.
By the way, Ras-1, one of the “target cells of Ras” is a serine/threonine protein kinase present in a cytoplasm, and the activity is induced by interaction with the GTP binding Ras. The activated Raf-1 phosphorylates MEK (MAPK/ERK kinase), and then MEK phosphorylates ERK to transmit signals into a nucleus (Daum, G., et al., (1994) Trends Biochem. Sci. 19, 474–480; Avruch, J., et al., (1994) Trends Biochem. Sci. 19, 279–283).
In order to elucidate such an intracellular signal transduction system of Raf-1, a method of selectively inhibiting the function of Ras or Raf-1 has been utilized (deVries-Smits, A. M., et al., (1992) Nature 357, 602–604). These studies include inhibition of the Ras function with a Raf-1 mutant free from a kinase activity (Kolch, W., et al., (1991) Nature 349, 426–428), inhibition of a Raf-1 kinase with an antibody bound to a kinase domain of Raf-1 (Kolch, W., et al., (1996) Oncogene 13, 1305–1314) and the like.
However, these inhibitors do not specifically inhibit a specific part of a signal transduction system with Ras or Raf-1, but inhibit many functions such as a function of binding to Ras, a kinase function and the like simultaneously and diversely. Accordingly, a signal transduction system to be inhibited cannot be specified. Thus, individual specific mechanisms of a signal transduction system could not be clarified satisfactorily.
Consequently, the development of a molecular seed capable of specifically inhibiting the binding of Ras to Raf-1 has become important for clarifying the role of the signal transduction system.
At present, a downstream signaling pathway of Ras has not been completely clarified. When such a molecular seed is developed, it is possible to elucidate the signaling pathway in which Ras participates using a molecular seed capable of specifically inhibiting some specific routes and clarify the signaling pathway with target proteins of Ras in detail. In addition, it is possible to control the intracellular signal transduction. Consequently, various diseases in which the intracellular signal transduction participates, such as tumors and the like, can be treated and prevented.
Meanwhile, the structural analysis of the “target proteins of Ras” in the intracellular signaling pathway in which Ras participates has been conducted. It has been known that the Ras binding domain (RBD) of Raf-1 is located from 51 to 131 residues in the N-terminus of Raf-1 (Vojtek, A. B., et al., (1993) Cell 74, 205–214; Chuang, E., et al., (1994) Mol. Cell. Biol. 14, 5318–5325).
Further, nucleic acid molecular seeds (aptamers), such as an RNA, a DNA and the like, having a high affinity for a certain target, such as proteins, have been isolated by “in vitro selection” methods (Ellington, A. D., et al., (1990) Nature 346, 818–822; Tuerk, C., et al., (1990) Science 249, 505‥510) (Bock, L. C., et al., (1992) Nature 355, 564–566; Qiu Qiu, Y. L., et al., (1994) Nucleic Acids Res. 22, 5229–5234; Gal. S. W., et al., (1998) Eur. J. Biochem. 252, 553–562; Bell, S. D., et al., (1998) J. Biol. Chem. 273, 14309–14314). Therefore, there is a possibility that an RNA specifically bound to Raf-1 is obtained by applying this method to Raf-1, while the interaction with an RNA is unknown.